Neutrophil chemotactic lymphokine, and method for the diagnosis of drug hypersensitive granulocytopenia using the same

ABSTRACT

The invention provides a neutrophil chemotactic lymphokine having an isoelectric point at 6.8-7.0, a cell line producing the lymphokine, and an agent for diagnosing drug-induced granulocytopenia, which comprises the lymphokine. When the diagnostic agent according to the invention is used for a patient, whether the patient has a possibility that drug-induced granulocytopenia may be caused or not can be diagnosed prior to the administration of a drug. Therefore, the patient can be safely treated without causing any side effect.

TECHNICAL FIELD

The present invention relates to a novel neutrophil chemotacticlymphokine and a diagnostic agent for diagnosing an attack ofdrug-induced granulocytopenia comprising this lymphokine.

BACKGROUND ART

Granulocytopenia refers to a condition that the number of granulocytesin peripheral blood is decreased, and is a concept includingagranulocytosis marked by severe decrease or loss in granulocytes andaccompanied by a grave condition. Of these, the agranulocytosis has ahigh mortality due to infectious diseases.

As causes of the granulocytopenia, have been known the attack attendingon other various diseases, administration of a drug, and the like. Ofthese, the granulocytopenia caused by the drug includes drug-inducedgranulocytopenia. The attack of the drug-induced granulocytopenia cannotbe predicted prior to the administration of the drug under thecircumstances. Further, no drug for treating the drug-inducedgranulocytopenia after the attack has been known, and so its treatmentonly depends on stopping the administration of a causative drug underthe circumstances.

Accordingly, it is an object of the present invention to provide adiagnostic agent capable of predicting an attack of drug-inducedgranulocytopenia.

DISCLOSURE OF THE INVENTION

In view of the foregoing circumstances, the present inventors have paidattention to granulocyte chemotactic factors, particularly, neutrophilchemotactic lymphokines and carried out a varied investigation as totheir diversity and specificity. As a result, it has been found that aplurality of subspecies different in isoelectric point from one otherexist in the neutrophil chemotactic lymphokines. The inventors havesucceeded in isolating a novel lymphokine not reported heretoforetherefrom. It has also been found that there is a correlation betweenthe chemotaxis of a neutrophil by such a novel lymphokine and the attackof drug-induced granulocytopenia, namely, an individual having aneutrophil low in chemotactic capability to the lymphokine is attackedby drug-induced granulocytopenia, and that the same correlation asdescribed above is also present in a GM-CSF (granulocyte-macrophagecolony-stimulating factor), thus leading to completion of the presentinvention.

According to the present invention, there are thus provided a neutrophilchemotactic lymphokine having an isoelectric point at 6.8-7.0 and a cellline which produces such a lymphokine.

According to the present invention, there are also provided an agent anda kit for diagnosing drug-induced granulocytopenia, which comprises thelymphokine described above.

According to the present invention, there are further provided an agentand a kit for diagnosing drug-induced granulocytopenia, which comprisesa GM-CSF.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 diagrammatically illustrates a relationship between isoelectricfocusing fractions of CFR-24 culture solution and neutrophil chemotacticactivity,

FIG. 2 diagrammatically illustrates relationships between chemotaxisindices (CI) of a neutrophil to NCL-4 and GM-CSF and specimens, and

FIG. 3 diagrammatically illustrates relationships between expressionrates of CD11b in a neutrophil by NCL-4 and GM-CSF and specimens.

BEST MODE FOR CARRYING OUT THE INVENTION

The neutrophil chemotactic lymphokine (hereinafter referred to as NCL-4)having an isoelectric point at 6.8-7.0 according to the presentinvention can be obtained from a T cell line obtained by transforming,for example, a normal T cell with a virus.

Here, the NCL-4 producing T cell line can be obtained by coculturing,for example, a normal human mononuclear leukocyte together with a humanadult T cell leukemia cell line (for example, MT2) and establishing a Tcell line transformed with a virus produced by the leukemia cell line.

In order to collect NCL-4 from this virus-transformed T cell line, it isonly necessary to culture this cell line, conduct isoelectric focusingof the culture solution thereof using neutrophil chemotactic capabilityas an index and collect a fraction having an isoelectric point of6.8-7.0.

The NCL-4 thus obtained has the following properties:

(1) having neutrophil chemotactic capability;

(2) having an isoelectric point (pI) of 6.8-7.0;

(3) increasing neither intracellular calcium ion concentration nor cellmembrane potential;

(4) inhibiting apoptosis (decrease in CD16);

(5) having the ability to express CD11b;

(6) having no affinity for heparin; and

(7) having a molecular weight of 50 kd to 80 kd (as measured by gelfiltration).

The GM-CSF useful in the practice of the present invention is a sort oflymphokine and has been known as a hematopoietic regulatory factor invivo, but has not been known to be used as a diagnostic agent fordrug-induced granulocytopenia.

The GM-CSF itself used in the present invention is known, can beprepared in accordance with a method known per se in the art (forexample, The Journal of Immunology, Vol. 137, P. 3584 (1986), etc.) andis also commercially available.

In order to diagnose drug-induced granulocytopenia using the diagnosticagent according to the present invention, which comprises the NCL-4 orGM-CSF, the reactivity of a neutrophil of a patient, to which a drugwill be administered, may be determined in the presence of thediagnostic agent according to the present invention. As a result, it maybe diagnosed that the patient has a high possibility of being attackedby drug-induced granulocytopenia if the reactivity of the neutrophil ofthe patient is low.

The reactivity of the neutrophil can be determined or confirmed inaccordance with a method known per se in the art. For example, it can beperformed by a method making use of the chemotaxis or of the expressionrate of CD11b (The Journal of Cell Biology, Vol. 120, No. 2, p. 545(1993)) of the neutrophil as an index.

According to the testing method of neutrophil chemotaxis, the reactivitycan be determined in accordance with the conventional means except thatthe diagnostic agent according to the present invention is used as achemotactic factor. Examples of such a testing method of neutrophilchemotaxis include a micropore filter method and an agarose method(Respiration, Vol. 4, No. 10, p. 1221 (1985); Journal of LeukocyteBiology, Vol. 51, p.617 (1992), etc.).

The result of the test of neutrophil chemotaxis is preferably evaluated,for example, by using chemotaxis index (CI value; chemotactic capabilityto the diagnostic agent according to present invention/chemotacticcapability to a control) as an index.

Further, the measurement of the expression rate of CD11b may also beconducted in a method known per se in the art, and can be easilyperformed by usual immunological analysis or assay making use of aspecific antibody thereof. Incidentally, the anti-CD11b antibody can beprepared in accordance with a method known per se in the art or is alsocommercially available.

The object of the diagnostic agent according to the present invention ispreferably a patient to which a drug already reported to causedrug-induced granulocytopenia or a drug having the possibility thereofwill be administered.

No particular limitation is imposed on the preparation form of thediagnostic agent according to the present invention so far as the agentcomprises the above-described NCL-4 or GM-CSF. For example, the agentmay be provided as a solution with the NCL-4 or GM-CSF dissolved in abuffer solution. The diagnostic agent according to the present inventionmay also be used as a kit for a clinical test in combination with otherreagents, buffers, necessary instruments and the like. Upon diagnosis,the diagnostic agent comprising NCL-4 and the diagnostic agentcomprising GM-CSF are separately used to collectively judge the resultsthus obtained, whereby the diagnosis can be made with higher precision.

EXAMPLES

The present invention will hereinafter be described in detail by thefollowing Examples. However, the present invention is not limited tothese examples.

Example 1

Establishment of NCL-4 producing cell line:

Peripheral blood mononuclear leukocytes (MNL, 100 cells/well) werecocultured with an irradiated (20,000 rad) human adult T cell leukemiacell line (MT2). This culture was performed in an RPMI-1640 solutioncontaining 10% fetal calf serum (FCS) and 2 mM L-glutamine in a 96-wellplate under conditions of 37° C. and moistened air containing 5% CO₂. Ahalf of the medium was changed every 3 days. After 1-3 months, thegrowth of the cells became stable, and the expression of an ATL-antigenwas observed in the greater part thereof. As the result of an HLA test(typing), it was confirmed that the cultured cells were derived from theperipheral blood leukocytes of the donor and not from the MT-2 cells.This cell line was cloned at a concentration of 0.5-1/well withirradiated (20,000 rad) autogenous MNL "filler" cells.

The cell line thus established was stored in an RPMI-1640 solutioncontaining 10% FCS. In order to obtain medium conditions for the T cellline, the cells (2×10⁵ cells/ml) were cultured over 3 days at 37° C. inmoistened air containing 5% CO₂ using a serum-free Iscove's-modifiedDulbecco's medium (IMDM) containing 2 mM L-glutamine, a 25 mM Hepesbuffer solution and 5×10⁻⁵ M 2-mercaptoethanol.

Whether the established T cell line produces a neutrophil chemotacticfactor or not was investigated by testing the culture solution thereofas to neutrophil chemotaxis (according to a method which will bedescribed subsequently).

The culture solution of the MT-2 cells did not exhibit neutrophilchemotaxis. Some of the established T cell lines exhibited chemotaxis.In particular, CFR-24 of these T cell lines most notably exhibitedneutrophil chemotaxis (FIG. 1). Therefore, CFR-24 was used in productionof NCL-4 in the following examples. The phenotype of CFR-24 wasanalyzed. As a result, it was found that most of them had T cell markerssuch as CD2 (94.8%), CD3 (52.8%), CD4 (83.6%) and CD5 (87.9%), and someof them had CD8 (16.1%). On the other hand, CFR-24 had neithergranulocyte/macrophage markers nor B cell markers such as CD16 (11.0%)and Leu7 (1.7%). Further, CFR-24 is considered to be in an activatedstate because it had CD25 (93.02%) and HLA-DR (100%). It was found fromthe above that CFR-24 belongs to a helper T cell lineage in activatedform.

Example 2

Isoelectric Focusing of Neutrophil Chemotactic Lymphokines

Preparative isoelectric focusing was conducted using a carrier ampholyte(Bio-Lyte 3/10; Bio-Rad in Richmond) and a Rotofor system (Bio-Rad inRichmond). The CFR-24 cultured medium, which had been fully dialyzedagainst 1% glycine, was used in an amount of 50 ml. Twenty fractionswere collected by the electrofocusing and fully dialyzed against aphosphate buffer solution (pH: 7.4), thereby investigating theneutrophil chemotaxis of the individual fractions.

As a result, it was recognized 4 fractions by which a neutrophil of ahealthy person clearly exhibited chemotaxis (FIG. 1). Of the fourfractions, fractions separately having a pI of 4-4.4, a pI of 4.6-5.0, apI of 5.9-6.2 and a pI of 6.8-7.0 were designated NCL-1, NCL-2, NCL-3and NCL-4, respectively.

Example 3

Properties of NCL-4

(1) Materials

Blood was collected by a vacuum blood collecting tube containingheparin. Separation of granulocytes was performed in the followingmanner. Added to 20 ml of a 3% dextran-PBS solution were 10 ml of theheparinized blood, and the mixture was fully blended and left at restfor 15 minutes, thereby obtaining a supernatant containing granulocytes.The supernatant was applied on 15 ml of a Ficoll pack (Pharmacia) andcentrifuged at 550×g for 30 minutes, thereby dividing it into alymphocyte layer and a granulocyte sediment. In order to removeerythrocytes mixed therein from the granulocyte sediment, 10 ml of coldwater were first added, and the mixture was left over for 30 seconds tohemolyze the erythrocytes. Then, 10 ml of PBS concentrated twice wereadded to return to isotonicity. Further, 15 ml of a Hanks' solution wereadded, and the mixture was then centrifuged at 550×g for 5 minutes. Thesediment was washed again with 15 ml of a Hanks' solution, therebyobtaining granulocytes. The granulocytes were suspended at aconcentration of 2×10⁶ cells/ml in an RPMI solution containing 10% FBSand used in a test of chemotactic capability and determination of CD16.

(2) Determination Methods

The molecular weight of NCL-4 was determined in the following manner.After a column of Cellulofine GCL-1000 (2.5×40 cm, Seikagaku Kogyo Co.,Ltd.) was equilibrated with a 0.05 M Tris-HCl/0.1 M KCl solution (pH:7.5), the NCL-4 was applied to fractionate every 3 ml, therebyidentifying the molecular weight of NCL-4 by determining the chemotacticactivity thereof.

Its affinity for heparin was identified by mixing NCL-4 (500 μl) and anAffinity Gel Heparin (Bio-Rad, 50 μl) at room temperature for 90minutes, and then centrifuging the mixture to determine the chemotacticresultant supernatant and of a liquid dissolved out of the sediment by atreatment with 2M NaCl.

Test of Chemotactic Activity

A 24-well Transwell having a pore size of 3 μm manufactured by CoasterCo. was used. Added to outer wells thereof were 600 Al of a chemotacticfactor [GM-CSF, TNF, 10 ng/ml (Genzyme); NCL-4, 100 μl/600 μl; IL-8, 3ng/ml (Otuka Pharmaceutical Co., Ltd.); C5a, 3 ng/ml (Sigma); fMLP, 10nM (Sigma); or LTB4, 100 pg/ml (Sigma)], and 100 μl of the granulocytesuspension were added to inner wells thereof, thereby reacting them for2 hours in a CO² incubator at 37° C. After completion of the reaction,the inner wells were removed, and 0.5 ml of cold 0.1% BSA-0.1% NaN₃ -PBSwere added to the outer wells. After fully stirring, the number of cellsin each of the outer wells was counted by a flow cytometer (SpectrumIII). As a control, 0.05% BSA-PBS was used, and the above factors werediluted with this solution. The chemotactic capability was expressed interms of a chemotaxis index which was obtained by dividing the number ofcells migrated by the chemotactic factor by the number of cells migratedby the control.

Effect of inhibiting decrease in CD16 (effect of inhibiting apoptosis)

Placed in a 96-well culture dish (Corning) were 50 μl of the granulocytesuspension and 50 μl of a stimulating factor, thereby conducting culturefor 24 hours at 370 in a CO₂ incubator. Thereafter, 0.5 ml of 1%BSA-0.1% NaN₃ -PBS were added, and the mixture was fully stirred andthen centrifuged (1,500 rpm, 5 minutes). After a supernatant was suckedout, and the sediment was fully stirred, 20 μl of an anti-CD16 antibody(Becton Dickinson) were added to conduct a reaction for 1 hour in icewater. After completion of the reaction, 1.0 ml of 1% BSA-0.1% NaN₃ -PBSwas added, and the mixture was fully stirred and then centrifuged (1,500rpm, 5 minutes). After a supernatant was sucked out, and the sedimentwas fully stirred, 0.5 ml of 1% BSA-0.1% NaN₃ -PBS were added to suspendthe sediment, thereby analyzing the suspension by means of a flowcytometer (Spectrum III).

Production of IL-8

Placed in a 96-well culture dish (Corning) were 50 μl of the granulocytesuspension and 50 μl of a stimulating factor, thereby conducting culturefor 24 hours at 370 in a CO₂ incubator. Thereafter, 0.1 ml of 1%BSA-0.1% NaN₃ -PBS were added, the mixture was fully stirred and thencentrifuged (1,500 rpm, 5 minutes), and 0.1 ml of a supernatant weretaken out, thereby determining IL-8 by enzyme immunoassay.

Expression of CD11b

Heparinized blood was poured in 50-μl portions into tubes, and 20 μl of0.05% BSA-PBS and a stimulating factor were added to each of the tubes,thereby conducting a reaction at 37° C. for 20 minutes. After completionof the reaction, the reaction mixture was washed with 1 ml of cold 0.1%BSA-0.1% NaN₃ -PBS, and 20 μl of an anti-CD11b antibody (BectonDickinson) were added to conduct a reaction for 1 hour in ice water.After completion of the reaction, the reaction mixture was hemolyzedwith a hemolytic reagent and centrifuged. The sediment was thensuspended in 0.5 ml of cold 0.1% BSA-0.1% NaN₃ -PBS, thereby analyzingthe suspension by means of a flow cytometer (Spectrum III).

Test for Measuring the Concentration of Calcium

Added to 200 μl of heparinized blood were 20 μl of 0.09 mM Fluo-3 AM(Dojindo), thereby loading the cells with Fluo-3 over 30 minutes at roomtemperature. Thereafter, the cells were hemolyzed, washed with a Hanks'solution and then suspended in 2 ml of 0.05% BSA-PBS. Added to 0.5 ml ofthe cell suspension were 5 μl of a stimulating factor, and analysis wasimmediately performed by means of a flow cytometer (Spectrum III).

Test for Measuring Membrane Potential

After 100 μl of heparinized blood were hemolyzed and washed with aHanks' solution, it was suspended in 1 ml of 0.05% BSA-PBS and reactedwith 1 μM Bis-oxanol (Mol. Prob) at room temperature for 30 minutes,thereby loading the cells with Bis-oxanol. Added to 0.5 ml of the cellsuspension were 50 μl of a stimulating factor, and after 2 minutes,analysis was performed by means of a flow cytometer (Spectrum III).

Concentrations of Cytokines in the NCL-4 Fraction

One milliliter of the NCL-4 fraction was taken out to separatelydetermine IL-lα, IL-1β, IL-6, IL-2, IL-8, TNF-α, GM-CSF, G-CSF, IFN-αand IFN-γ by enzyme immunoassay, and M-CSF by radioimmunoassay.

(3) Results

As shown in Table 1, NCL-4 has chemotactic activity, but does notincrease an intracellular Ca concentration and a cell membrane potentialunlike the already known chemotactic factors such as IL-8, C5a, fMLP andLTB4. NCL-4 has an effect of inhibiting decrease in CD16 (effect ofinhibiting apoptosis) though such an effect is not recognized in thealready known chemotactic factors such as IL-8, C5a, fMLP and LTB4.GM-CSF, G-CSF and TNF-α also have neutrophil chemotactic activity, butGM-CSF is different from NCL-4 in that GM-CSF does not induce theproduction of IL-8 from the neutrophil. As shown in Table 2, as theresult of determining various cytokines contained in the NCL-4 fraction,it was found that neither TNF-α nor G-CSF is contained in the NCL-4fraction, but GM-CSF and IL-2 are recognized. As shown in Table 1,however, IL-2 has no chemotactic activity. NCL-4 is also considered asubstance having the nature different from the known substances from theviewpoints of the respective molecular weights, isoelectric points andaffinity for heparin (Table 1).

                                      TABLE 1                                     __________________________________________________________________________                       NCL-4                                                                             GM-CSF                                                                             G-CSF                                                                             TNF-α                                                                       IL-8                                                                             IL-2                                                                             C5a fMLP                                                                             LTB4                         __________________________________________________________________________    Granulocyte chemotactic activity (CI)                                                            9.9 11.3 6.5  7.1                                                                              49.2                                                                             0.0                                                                              36.4                                                                              34.0                                                                             28.4                           Expression of CD11B (Δ Mean channel) 8.2 15.1 2.6 31.0 21.9 --                                                         43.4 45.0 --                   Increase in intracellular Ca (% positive) 0.0  0.0 0.0  0.0 59.0 --                                                          80.0 84.0 40.0                 Increase in membrane potential (% positive) 0.0  0.0 0.0 -- 13.0 --                                                          88.0 71.0 --                   Inhibition of apoptosis (% of inhibition) 38.2  42.9 -- 29.8  0.0 0.0                                                        0.0  0.0 --                    Production of IL-8 (pg/ml) 214    <20   -- 179    -- -- -- -- --                                                              Molecular weight (kb)                                                        50-80 22   19   36   6-8                                                      15    8.6  0.4  0.3                                                            Isoelectric point                                                            6.8-7.0 3.4-4.5 -- --                                                         8.6 -- -- -- --                Affinity for heparin None None -- -- Yes -- -- -- --                        __________________________________________________________________________     --: Not investigated.                                                    

                                      TABLE 2                                     __________________________________________________________________________    Various cytokines contained in the NCL-4 fraction                                   IL-1α                                                                       IL-1β                                                                        IL-6                                                                              IL-2                                                                              IL-8                                                                              TNF-α                                                                       CM-CSF                                                                             G-CSF                                                                             M-CSF                                                                             IFN-α                                                                       IFN-γ                      (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)                                                    (pg/ml) (pg/ml)                __________________________________________________________________________    NCL-4 <10 <20 <20 759 <20 <20 34   <20 <0.2                                                                              <20 <20                              Sensitivity to  10  20  20  50  20  20 20  20  0.2  20  20                    measurement                                                                 __________________________________________________________________________

Example 4

Test of Neutrophil Chemotaxis

The human peripheral bloods used in this experiment were provided frompatients and healthy persons unless expressly noted. Neutrophils wereisolated from the bloods collected by vacuum blood collecting tubescontaining heparin in accordance with the following process. Added to 20ml of a 3% dextran-PBS solution were 10 ml of the heparinized blood, andthe mixture was fully mixed and left at rest for 15 minutes, therebyobtaining a supernatant containing granulocytes. The supernatant wasapplied on 15 ml of a Ficoll pack (product of Pharmacia) and centrifugedat 550×g for 30 minutes, thereby dividing it into a lymphocyte layer anda neutrophil sediment. In order to remove erythrocytes from theneutrophil sediment, 10 ml of cold water were added, and the mixture wasleft over for 30 seconds to hemolyze the erythrocytes. Then, 10 ml ofPBS concentrated twice were added to return to isotonicity. Further, 15ml of a Hanks' solution were added, and the mixture was then centrifugedat 550×g for 5 minutes. The sediment was washed again with 15 ml of aHanks' solution, thereby obtaining neutrophils. The neutrophils thusobtained were suspended at a concentration of 2×10⁶ cells/ml in an RPMIsolution containing 10% FBS and used in a test of chemotaxis. The purityof the neutrophils was at least 95%, and their survival rate was also atleast 95%.

The chemotaxis was determined in accordance with the test of chemotacticactivity in Example 3(2) or by the following process.

A 48-well microchemotaxis chamber equipped with a polyvinylpyrrolidone-free Nuclepore filter having a pore size of 3 μm forneutrophils was used to conduct analysis. After the neutrophils (1-2×10⁶cells/ml) were incubated at 37° C. for 2 hours, cells adhered to thelower surface through the filter were stained with a Diff-Quick (TheGreen Cross Corporation) and counted through an optical microscope of 5high power field (hpf, 40×10). The measurement result as to theneutrophil chemotaxis was expressed as the mean number±SD of migratedcells/hpf in triplicate. In this result of the chemotaxis test, theneutrophil chemotaxis index of the patients was expressed as CI. The CIwas calculated out in accordance with the equation: (chemotacticactivity to NCL-4 or GM-CSF)/(chemotactic activity to PBS).

Example 5

Comparison of Patients Suffering From Drug-Induced Granulocytopenia WithHealthy Persons as to Neutrophil Chemotaxis

The neutrophil chemotaxis to the four neutrophil chemotactic factorsderived from CFR-24 of various patients and healthy persons wasinvestigated. The result is shown in Table 3.

Incidentally, the patients are patients suffering from congestive heartfailure, to which Arkin Z had been administered. Arkin Z is an agentuseful for treating heart failure and is known to cause agranulocytosis(about 0.3%).

                  TABLE 3                                                         ______________________________________                                                     Group of patients                                                                        Group of patients                                       attacked by not attacked by                                                   granulocytopenia granulocytopenia                                           ______________________________________                                        NCL-4                                                                           95% CI (upper limit < 4.20) 16/21 76.2% 12/48 25.0%                           99% CI (upper limit < 4.59) 17/21 81.0% 15/48 31.3%                           GM-CSF                                                                        95% CI (upper limit < 8.31)  8/10 80.0% 19/33 57.6%                           99% CI (upper limit < 9.36)  8/10 80.0% 20/33 60.6%                         ______________________________________                                    

As a result, with respect to the neutrophils of the groups of healthypersons and patients not attacked by granulocytopenia (patientssuffering from heart failure and not attacked by granulocytopenia uponthe administration of Arkin Z), chemotaxis was induced by the fourdifferent NCLs derived from CFR-24. With respect to the neutrophils ofthe group of patients attacked by granulocytopenia (patients sufferingfrom heart failure and attacked by granulocytopenia after theadministration of Arkin Z), however, chemotaxis was induced by NCL-1 andNCL-2, but not induced by NCL-3 and NCL-4. Therefore, the neutrophilchemotaxis to NCL-4 and the neutrophil chemotaxis to C5a werecomparatively analyzed between the group of the attacked patients andthe group of the unattacked patients using the index CI. As a result,with respect to C5a, there was no difference in the CI of theneutrophils among the three groups. On the other hand, with respect toNCL-4 and GM-CSF, a marked difference was recognized between the groupof the attacked patients and the group of the unattacked patients asshown in Table 3 and FIG. 2. More specifically, the CI to NCL-4 in thegroup of the attacked patients was smaller than 4.59 (the upper limit of99% conficence interval (CI) of the group of the attacked patients) in17 cases of 21 cases, while the CI to NCL-4 in the group of theunattacked patients was smaller than 4.59 in 15 cases of 48 cases.Besides, the CI to GM-CSF in the group of the attacked patients wassmaller than 9.36 (the upper limit of 99% confidence interval (CI) ofthe group of the attacked patients) in 8 cases of 10 cases, while the CIto GM-CSF in the group of the unattacked patients was smaller than 9.36in 20 cases of 33 cases.

From the above results, it can be diagnosed that any patient sufferingfrom heart failure and having a CI value to NCL-4 of 5 or smaller has apossibility of being attacked by granulocytopenia when Arkin Z is usedin treatment for heart failure. It can also be diagnosed that anypatient suffering from heart failure and having a CI value to GM-CSF of10 or smaller has a possibility of being attacked by granulocytopeniawhen Arkin Z is used in the treatment.

Example 6

Test of the Ability to Express CD11b

Heparinized blood collected by a vacuum blood collecting tube containingheparin was poured in 50-μl portions into tubes, and 20 μl of 0.05%BSA-PBS, NCL-4 or GM-CSF (10 ng/ml, Genzyme) were added to the tube,thereby conducting a reaction at 37° C. for 20 minutes. After completionof the reaction, the reaction mixture was washed with 1 ml of cold 0.1%BSA-0.1% NaN₃ -PBS, and 20 μl of an anti-CD11b antibody (BectonDickinson) were added to conduct a reaction for 1 hour in ice water.After completion of the reaction, the reaction mixture was hemolyzedwith a hemolytic reagent and centrifuged. The sediment was thensuspended in 0.5 ml of cold 0.1% BSA-0.1% NaN₃ -PBS, thereby analyzingthe suspension by means of a flow cytometer (Spectrum III). The resultsin the 0.05% BAS-PBS group was used as a control, and the expression ofCD11b was expressed in terms of % of the amount of CD11b more expressedthan the amount of CD11b expressed in the control.

The expression of CD11b was comparatively analyzed between the group ofpatients attacked by granulocytopenia (patients suffering from heartfailure and attacked by granulocytopenia after the administration ofArkin Z) and the group of patients not attacked by granulocytopenia(patients suffering from heart failure and not attacked bygranulocytopenia upon the administration of Arkin Z). The result isshown in Table 4.

                  TABLE 4                                                         ______________________________________                                                     Group of attacked                                                                        Group of unattacked                                     patients patients                                                           ______________________________________                                        NCL-4                                                                           95% CI (upper limit < 18.59) 7/10 70.0%  9/33 27.3%                           99% CI (upper limit < 20.85) 9/10 90.0% 10/33 30.3%                           GM-CSF                                                                        95% CI (upper limit < 11.34) 8/10 80.0% 12/33 36.4%                           99% CI (upper limit < 12.73) 9/10 90.0% 13/33 39.4%                         ______________________________________                                    

As a result, with respect to NCL-4 and GM-CSF, a marked difference wasrecognized between the group of the attacked patients and the group ofthe unattacked patients as shown in Table 4 and FIG. 3. Morespecifically, the expression of CD11b by NCL-4 in the group of theattacked patients was less than 20.85 (the upper limit of 99% confidenceinterval of the group of the attacked patients) in 9 cases of 10 cases,while the expression of CD11b by NCL-4 in the group of the unattackedpatients was less than 20.85 in 10 cases of 33 cases. Besides, theexpression of CD11b by GM-CSF in the group of the attacked patients wasless than 12.73 (the upper limit of 99% conficence interval of the groupof the attacked patients) in 8 cases of 10 cases, while the expressionof CD11b by GM-CSF in the group of the unattacked patients was less than12.73 in 13 cases of 33 cases.

From the above results, it can be diagnosed that any patient sufferingfrom heart failure and having low reactivity (chemotactic activity,expression of CD11b) to NCL-4 or GM-CSF has a possibility of beingattacked by granulocytopenia when Arkin Z is used in treatment for heartfailure.

INDUSTRIAL APPLICABILITY

When the diagnostic agent according to the present invention is used fora patient, whether the patient has a possibility that drug-inducedgranulocytopenia may be caused or not can be diagnosed prior to theadministration of a drug. Therefore, the patient can be safely treatedwithout causing any side effect.

What is claimed is:
 1. An isolated neutrophil chemotactic lymphokinehaving an isoelectric point at 6.8-7.0, wherein said lymphokine has theproperties of:a) increasing neither intracellular calcium ionconcentration nor cell membrane potential; b) inhibiting apoptosis bydecreasing CD16 expression; c) inducing expression of CD11b d) having noaffinity for heparin; and e) having a molecular weight of 50 kd to 80 kdas measured by gel filtration.
 2. A method of diagnosing whether asubject is susceptible to drug-induced granulocytopenia, which comprisesmeasuring reactivity of a neutrophil from said subject to GM-CSF or thelymphokine of claim
 1. 3. The method of diagnosing according to claim 2,wherein the reactivity of the neutrophil is determined by chemotaxis ofor the expression rate of CD11b of the neutrophil as an index ofreactivity.